Fragile X syndrome (FXS), a trinucleotide (CGG) repeat expansion disorder, is the leading heritable form of mental retardation, and is associated with a variety of learning disorders and behavioral problems in childhood. FXS is generally believed to arise when the CGG element of the fragile X mental retardation 1 (FMR1) gene expands beyond -200 repeats (full mutation), and the upstream promoter region and CGG repeat become methylated, the latter event leading to transcriptional silencing and the consequent failure to produce FMRI protein (FMRP). However, for males with premutation alleles (55-200 repeats) FMRI mRNA levels in peripheral blood leucocytes are significantly elevated, by as much as ten-fold for males with premutation alleles that exceed 100 repeats, and for males with full mutation alleles that remain unmethylated. These observations suggest that the FMR1 gene may be up-regulated. Identification of the reasons for the elevated mRNA levels is the first major objective of the proposed research. It has also been demonstrated that leucocytes from the majority of males with FMRI alleles that are fully expanded and (apparently) fully methylated continue to produce significant levels of FMR1 mRNA. This surprising result, which is not due to transcription from premutation alleles or a small fraction of fullyunmethylated alleles, suggests that the interplay between methylation and silencing is less direct than had been supposed. Identification of the means by which hypermethylated FMR1 genes sometimes escape silencing is the second major objective of the proposed research. These two basic observations have important ramifications for understanding and eventually treating FXS. Elucidation of the signals that regulate transcriptional activity (both for up-regulation and for silencing) will hopefully lead to molecular therapies that can modulate the expression of the endogenous FMR1 gene. Methods of approach will continue to be quantitative (fluorescence) RT-PCR, and will incorporate chromatin immunoprecipitation as an assay for acetylation status of FMR1-associated histones.